scholarly journals Crystal structure of recombinant tyrosinase-binding protein MtaL at 1.35 Å resolution

2016 ◽  
Vol 72 (3) ◽  
pp. 244-250 ◽  
Author(s):  
Xuelei Lai ◽  
Montserrat Soler-Lopez ◽  
Wangsa T. Ismaya ◽  
Harry J. Wichers ◽  
Bauke W. Dijkstra
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Agaricus Bisporus ◽  
Physiological Function ◽  
Carbohydrate Binding ◽  
Mushroom Tyrosinase ◽  
Binding Interface ◽  
Loop Regions ◽  
Carbohydrate Binding Site

Mushroom tyrosinase-associated lectin-like protein (MtaL) binds to matureAgaricus bisporustyrosinasein vivo, but the exact physiological function of MtaL is unknown. In this study, the crystal structure of recombinant MtaL is reported at 1.35 Å resolution. Comparison of its structure with that of the truncated and cleaved MtaL present in the complex with tyrosinase directly isolated from mushroom shows that the general β-trefoil fold is conserved. However, differences are detected in the loop regions, particularly in the β2–β3 loop, which is intact and not cleaved in the recombinant MtaL. Furthermore, the N-terminal tail is rotated inwards, covering the tyrosinase-binding interface. Thus, MtaL must undergo conformational changes in order to bind mature mushroom tyrosinase. Very interestingly, the β-trefoil fold has been identified to be essential for carbohydrate interaction in other lectin-like proteins. Comparison of the structures of MtaL and a ricin-B-like lectin with a bound disaccharide shows that MtaL may have a similar carbohydrate-binding site that might be involved in glycoreceptor activity.

scholarly journals Latent and activeabPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal

2014 ◽  
Vol 70 (9) ◽  
pp. 2301-2315 ◽  
Author(s):  
Stephan Gerhard Mauracher ◽  
Christian Molitor ◽  
Rami Al-Oweini ◽  
Ulrich Kortz ◽  
Annette Rompel
Keyword(s):  
Crystal Structure ◽  
Single Crystal ◽  
Agaricus Bisporus ◽  
Crystal Packing ◽  
Active Form ◽  
Activation Mechanism ◽  
Mushroom Tyrosinase ◽  
Polyphenol Oxidases ◽  
Highly Active ◽  
Eukaryotic Organisms

Tyrosinases, bifunctional metalloenzymes, catalyze the oxidation of monophenols ando-diphenols too-quinones, the precursor compounds of the brown-coloured pigment melanin. In eukaryotic organisms, tyrosinases are expressed as latent zymogens that have to be proteolytically cleaved in order to form highly active enzymes. This activation mechanism, known as the tyrosinase maturation process, has scientific and industrial significance with respect to biochemical and technical applications of the enzyme. Here, not only the first crystal structure of the mushroom tyrosinaseabPPO4 is presented in its active form (Ser2–Ser383) and in its 21 kDa heavier latent form (Ser2–Thr545), but furthermore the simultaneous presence of both forms within one single-crystal structure is shown. This allows for a simple approach to investigate the transition between these two forms. IsoformabPPO4 was isolated and extensively purified from the natural source (Agaricus bisporus), which contains a total of six polyphenol oxidases (PPOs). The enzyme formed crystals (diffracting to a resolution of 2.76 Å) owing to the employment of the 6-tungstotellurate(VI) salt (Na6[TeW6O24]·22H2O) as a cocrystallization agent. Two of these disc-shaped Anderson-type polyoxoanions [TeW6O24]6−separate two asymmetric units comprising one crystallographic heterodimer ofabPPO4, thus resulting in very interesting crystal packing.

scholarly journals Crystal structure of EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

2014 ◽  
Vol 70 (a1) ◽  
pp. C256-C256
Author(s):  
Beatriz Trastoy
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Igg Antibodies ◽  
Human Pathogens ◽  
X Ray ◽  
Effector Functions ◽  
Encounter Complex ◽  
Key Enzyme

In order to evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.

scholarly journals Molecular Modeling of Lectin-Like Protein fromAcacia farnesianaReveals a Possible Anti-Inflammatory Mechanism in Carrageenan-Induced Inflammation

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Vanessa Erika Ferreira Abrantes ◽  
Bruno Anderson Matias da Rocha ◽  
Raphael Batista da Nóbrega ◽  
José Caetano Silva-Filho ◽  
Claudener Souza Teixeira ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Carbohydrate Binding ◽  
Tertiary Structures ◽  
Acacia Farnesiana ◽  
Inflammatory Mechanism ◽  
Loop Regions ◽  
Anti Inflammatory ◽  
Chitin Binding Protein ◽  
Legume Lectins ◽  
Carbohydrate Binding Site

Acacia farnesianalectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin fromPhaseolus vulgaris(PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through structural comparison with anti-inflammatory legume lectins. The AFAL model was obtained by molecular modeling and molecular docking with glycan and carrageenan were performed to explain the AFAL structural behavior and biological activity.Pisum sativumlectin was the best template for molecular modeling. The AFAL structure model is folded as aβsandwich. The model differs from template in loop regions, number ofβstrands and carbohydrate-binding site. Carrageenan and glycan bind to different sites on AFAL. The ability of AFAL binding to carrageenan can be explained by absence of the sixthβ-strand (posteriorβsheets) and twoβstrands in frontal region. AFAL can inhibit pathway inflammatory process by carrageenan injection by connecting to it and preventing its entry into the cell and triggers the reaction.

Receptor engagement by viral interleukin-6 encoded by Kaposi sarcoma–associated herpesvirus

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3042-3049 ◽  
Author(s):  
Yoshiyasu Aoki ◽  
Masashi Narazaki ◽  
Tadamitsu Kishimoto ◽  
Giovanna Tosato
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Interleukin 6 ◽  
Kaposi Sarcoma ◽  
Enzyme Linked Immunosorbent Assay ◽  
Homologous Region ◽  
Binding Interface ◽  
Surface Resonance ◽  
The Common ◽  
Common Signal

Abstract Receptor usage by viral interleukin-6 (vIL-6), a virokine encoded by Kaposi sarcoma– associated herpesvirus, is an issue of controversy. Recently, the crystal structure of vIL-6 identified vIL-6 sites II and III as directly binding to glycoprotein (gp)130, the common signal transducer for the IL-6 family of cytokines. Site I of vIL-6, however, comprising the outward helical face of vIL-6, where human IL-6 (hIL-6) would interact with the specific α-chain IL-6 receptor (IL-6R), is accessible and not occupied by gp130. This study examined whether this unused vIL-6 surface is available for IL-6R binding. By enzyme-linked immunosorbent assay, vIL-6 bound to soluble gp130 (sgp130) but not to soluble IL-6R (sIL-6R). Using plasmon surface resonance, vIL-6 bound to sgp130 with a dissociation constant of 2.5 μM, corresponding to 1000-fold lower affinity than that of hIL-6/sIL-6R complex for gp130. sIL-6R neither bound to vIL-6 nor affected vIL-6 binding to gp130. In bioassays, vIL-6 activity was neutralized by 4 monoclonal antibodies (mAbs) recognizing a domain within vIL-6 site I, mapped to the C-terminal part of the AB-loop and the beginning of helix B. The homologous region in hIL-6 participates in site I binding to IL-6R. In addition, binding of vIL-6 to sgp130 was interfered with specifically by the 4 neutralizing anti–vIL-6 mAbs. Based on the vIL-6 crystal structure, the vIL-6 neutralizing mAbs map outside the binding interface to gp130, suggesting that they either produce allosteric changes or block necessary conformational changes in vIL-6 preceding its binding to gp130. These results document that vIL-6 does not bind IL-6R and suggest that conformational change may be critical to vIL-6 function.

scholarly journals Crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica

2019 ◽  
Vol 75 (3) ◽  
pp. 197-204
Author(s):  
Farha Khan ◽  
Kaza Suguna
Keyword(s):  
Crystal Structure ◽  
Entamoeba Histolytica ◽  
Intracellular Transport ◽  
Golgi Body ◽  
Carbohydrate Binding ◽  
Type I ◽  
Legume Lectin ◽  
Species Specific ◽  
Carbohydrate Binding Site

ERGIC-53-like proteins are type I membrane proteins that belong to the class of intracellular cargo receptors and are known to be indispensable for the intracellular transport of glycoproteins. They are implicated in transporting glycoproteins between the endoplasmic reticulum and the Golgi body. The crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica has been determined at 2.4 Å resolution. Although the overall structure of the domain resembles those of its mammalian and yeast orthologs (ERGIC-53 and Emp46, respectively), there are significant changes in the carbohydrate-binding site. A sequence-based search revealed the presence of several homologs of ERGIC-53 in different species of Entamoeba. This is the first report of the structural characterization of a member of this class of proteins from a protozoan and serves to further knowledge and understanding regarding the species-specific differences.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Laetitia Seguin ◽  
Soline Odouard ◽  
Francesca Corlazzoli ◽  
Sarah Al Haddad ◽  
Laurine Moindrot ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Survival ◽  
Small Gtpases ◽  
Molecular Signature ◽  
Carbohydrate Binding ◽  
Pancreatic Cancers ◽  
Galectin 3 ◽  
Pharmacologic Inhibition

AbstractRecently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and β1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles’ heel for a significant and unique subset of GBM patients.

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